Optimized total RNA isolation from bovine sperm with enhanced sperm head lysis.

Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities.

Comparison of the developmental competence of in vitro-produced mouse embryos cultured under 5 versus 2% O2 with in vivo-derived blastocysts.

PURPOSE: The prevalence of infertility in Canada has substantially increased over 30 years, and plateaued success rates of culture systems warrant further optimization for transfer outcomes. In clinical programs, embryos commonly undergo extended culture under 5% O2 until the blastocyst stage. The aim of this study is to characterize the developmental competence and stress-related responses of embryos cultured under 5 versus 2% O2 in comparison to in vivo-derived blastocysts. We hypothesized 2% O2 compromises developmental competence through altered embryonic stress responses and induction of apoptosis-related genes relative to those cultured under 5% O2 and in vivo-derived blastocysts. METHODS: Quantitative measures of development and relative expressions of a cohort of stress-related genes in CD1 mouse zygotes cultured to blastocysts under 5 or 2% O2 were compared to in vivo-derived embryos. Apoptotic responses were evaluated using an immunofluorescence assay for Caspase-3. RESULTS: The mean percentage of blastocysts developed, and total cell number of embryos derived in vivo or cultured under 5% O2 was significantly higher than those cultured under 2% O2. Blastocyst expansion was greatest in embryos cultured under 5% O2. Stress response genes were significantly upregulated in embryos cultured under 2% O2, and expression of antioxidant-related genes was significantly lower in cultured versus in vivo-derived embryos. Caspase-3 immunofluorescence was significantly higher in cultured embryos versus in vivo-derived embryos. CONCLUSION: We inferred that 5% O2 systems better approximate physiologic oxygen availability for culture of mouse embryos, warranting re-evaluation of culturing embryos under threshold or sub-physiologic oxygen concentrations during clinical IVF programs.

Melatonin improves post-thaw sperm quality after mild testicular heat stress in rams.

The objective was to determine effects of slow-release melatonin on post-thaw sperm quality in rams exposed to mild testicular heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams were randomly and equally allocated to receive either 36 mg melatonin in 1 ml corn oil or 1 ml corn oil injected subcutaneously (SQ); 15 day later, all rams had HS for 96 h (start of HS = start of Week 0). Semen was collected before HS and once weekly from Weeks 1 to 7, extended in Steridyl CSS One Step, held at 5°C for ~3 h, loaded into 0.5 ml straws, held 5 cm above liquid nitrogen for 10 min and then plunged. Computer assisted semen analysis (CASA) was conducted on frozen-thawed sperm. There were group and week effects for total and progressive motility (p < .001), plus group and week effects and group*week interactions (p < .001) for post-thaw total abnormalities, acrosome integrity, post-thaw sperm DNA fragmentation index (DFI) and high mitochondrial membrane potential (HMMP). Post-thaw sperm total and progressive motility, acrosome integrity and HMMP were higher (p < .05) in melatonin versus control groups from Weeks 1 to 7, and the melatonin group reached baseline level (pre-heat stress) at Week 7 (75.79 ± 0.96, 65.48 ± 1.51, 75.00 ± 0.89 and 67.00 ± 1.06, respectively; mean ± SEM). Conversely, post-thaw sperm total abnormalities and DFI were lower (p < .05) in melatonin versus control, and both reached baseline at Week 7 in the melatonin group (26.00 ± 0.57 and 5.66 ± 0.17, respectively). Coiled tails, distal midpiece reflexes, distal cytoplasmic droplets, ruffled acrosomes, bowed midpieces, pyriform heads and knobbed acrosomes were the most common abnormalities in both groups, with lower percentages in melatonin-treated rams. Results supported our hypothesis that HS reduces post-thaw sperm quality, and that melatonin lessens those reductions, manifested by significantly better total and progressive motility, acrosome integrity and HMMP, and fewer sperm total abnormalities and DFI.

Characterization of the Testis-Specific Angiotensin Converting Enzyme (tACE)-Interactome during Bovine Sperm Capacitation.

A comprehensive understanding of molecular and biochemical changes during sperm capacitation is critical to the success of assisted reproductive technologies. We reported involvement of the testis-specific isoform of Angiotensin Converting Enzyme (tACE) in bovine sperm capacitation. The objective of this study was to characterize the tACE interactome in fresh and heparin-capacitated bovine sperm through immunoprecipitation coupled with mass spectrometry. These interactions were validated by co-localization of tACE with beta-tubulin as an identified interactome constituent. Although interactions between tACE and several proteins remained unchanged in fresh and capacitated sperm, mitochondrial aldehyde dehydrogenase 2 (ALDH2), inactive serine/threonine protein-kinase 3 (VRK3), tubulin-beta-4B chain (TUBB4B), and tubulin-alpha-8 chain (TUBA8) were recruited during capacitation, with implications for cytoskeletal and membrane reorganization, vesicle-mediated transport, GTP-binding, and redox regulation. A proposed tACE interactional network with identified interactome constituents was generated. Despite tACE function being integral to capacitation, the relevance of interactions with its binding partners during capacitation and subsequent events leading to fertilization remains to be elucidated.

Prepubertal nutritional modulation in the bull and its impact on sperm DNA methylation.

Enhanced pre-pubertal nutrition in Holstein bulls increased reproductive hormone production and sperm production potential with no negative effects on sperm quality. However, recent trends in human epigenetic research have identified pre-pubertal period to be critical for epigenetic reprogramming in males. Our objective was to evaluate the methylation changes in sperm of bulls exposed to different pre-pubertal diets. One-week-old Holstein bull calves (n = 9), randomly allocated to 3 groups, were fed either a high, medium or low diet (20%, 17% or 12.2% crude protein and 67.9%, 66% or 62.9% total digestible nutrients, respectively) from 2 to 32 weeks of age, followed by medium nutrition. Semen collected from bulls at two specific time points, i.e. 55-59 and 69-71 weeks, was diluted, cryopreserved and used for reduced representation bisulfite sequencing. Differential methylation was detected for dietary treatment, but minimal differences were detected with age. The gene ontology term, "regulation of Rho protein signal transduction", implicated in sperm motility and acrosome reaction, was enriched in both low-vs-high and low-vs-medium datasets. Furthermore, several genes implicated in early embryo and foetal development showed differential methylation for diet. Our results therefore suggest that sperm epigenome keeps the memory of diet during pre-pubertal period in genes important for spermatogenesis, sperm function and early embryo development.

Testis-Specific Isoform of Na+-K+ ATPase and Regulation of Bull Fertility.

An advanced understanding of sperm function is relevant for evidence-based male fertility prediction and addressing male infertility. A standard breeding soundness evaluation (BSE) merely identifies gross abnormalities in bulls, whereas selection based on single nucleotide polymorphisms and genomic estimated breeding values overlooks sub-microscopic differences in sperm. Molecular tools are important for validating genomic selection and advancing knowledge on the regulation of male fertility at an interdisciplinary level. Therefore, research in this field is now focused on developing a combination of in vitro sperm function tests and identifying biomarkers such as sperm proteins with critical roles in fertility. The Na+-K+ ATPase is a ubiquitous transmembrane protein and its α4 isoform (ATP1A4) is exclusively expressed in germ cells and sperm. Furthermore, ATP1A4 is essential for male fertility, as it interacts with signaling molecules in both raft and non-raft fractions of the sperm plasma membrane to regulate capacitation-associated signaling, hyperactivation, sperm-oocyte interactions, and activation. Interestingly, ATP1A4 activity and expression increase during capacitation, challenging the widely accepted dogma of sperm translational quiescence. This review discusses the literature on the role of ATP1A4 during capacitation and fertilization events and its prospective use in improving male fertility prediction.

Ouabain-induced activation of phospholipase C zeta and its contributions to bovine sperm capacitation.

The sperm-derived oocyte activating factor, phospholipase C zeta (PLC ζ), is the only PLC isoform reported in cattle. The objectives were to (1) localize PLC ζ in fresh and capacitated bovine sperm and (2) investigate the activation of PLC ζ during bull sperm capacitation and contributions of PLC activity to this process. We confirmed interaction of testis-specific isoform of Na/K-ATPase (ATP1A4) with PLC ζ (immunolocalization and immunoprecipitation) and tyrosine phosphorylation (immunoprecipitation) of PLC ζ (a post-translational protein modification commonly involved in activation of PLC in somatic cells) during capacitation. Furthermore, incubation of sperm under capacitating conditions upregulated PLC-mediated hyperactivated motility, tyrosine phosphoprotein content, acrosome reaction, and F-actin formation (flow cytometry), implying that PLC activity is enhanced during capacitation and contributing to these capacitation processes. In conclusion, we inferred that PLC ζ is activated during capacitation by tyrosine phosphorylation through a mechanism involving ATP1A4, contributing to capacitation-associated biochemical events.

Scrotal subcutaneous temperature is increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decrease sperm quality in bulls and rams.

Ruminant testes are ~2-6 °C below body temperature; increased testicular temperature reduces sperm motility and morphology. Our objective was to serially monitor scrotal subcutaneous temperatures during testicular heat stress and relate those to sperm quality. Two experiments were conducted, with temperature sensors surgically implanted in scrotal subcutaneous tissues recording temperatures every 15 min and semen collected and evaluated weekly. After an initial control interval, testicular temperature was increased. In Experiment 1, in two Angus bulls, whole-scrotum insulation for 96 h increased scrotal subcutaneous temperatures by ~2.0-2.5 °C (P < 0.05). Total and progressive motility decreased (P < 0.05) and reached a nadir at Week 3 (~20 and 10%, respectively). Furthermore, morphologically normal sperm and acrosome integrity also decreased significantly, reaching nadirs at Weeks 3 (15%) and 4 (34%). In Experiment 2, 10 Dorset rams were allocated randomly into two equal groups and either: 1) exposed to 28 °C ambient temperature and 30-34% RH for 8 h/d for 4 d; or 2) subjected to scrotal neck insulation that was applied and removed at the same time as the start and completion, respectively, of heat exposures in the other rams. Scrotal subcutaneous temperatures (monitored in three rams per group) were increased in response to whole-body heating (~0.8 °C, P < 0.05), but not significantly changed by scrotal neck insulation. Decreases in sperm quality were generally similar between treatments, with the most profound changes evident 4 wk after heat stress, with ~10 percentage point reductions in both total and progressive motility and ~10 and 20 percentage point decreases in morphologically normal sperm in rams with whole-body heating versus scrotal neck insulation, respectively. In conclusion, scrotal subcutaneous temperature was significantly increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decreased sperm motility and morphology in bulls and rams.

Role of Akt and mammalian target of rapamycin signalling in insulin-like growth factor 1-mediated cell proliferation in porcine Sertoli cells.

The critical role of insulin-like growth factor (IGF) 1 in promoting Sertoli cell proliferation invivo and invitro has been established, but its downstream signalling mechanisms remain unknown. In addition to mitogenic effects, a role for IGF1 in mediating cholesterol biosynthesis within testes has been implied. The aims of this study were to investigate the roles of: (1) phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signalling in IGF1-mediated Sertoli cell proliferation; and (2) IGF1 in mediating cholesterol biosynthesis in Sertoli cells. Primary cultures of Sertoli cells were prepared from 1-week-old porcine testes. On Day 3 of culture, Sertoli cells were treated with 300ng mL-1 IGF1, alone or in combination with inhibitors of IGF1 receptor (2µM picropodophyllotoxin), Akt (1µM wortmannin) or mTOR (200nM rapamycin). Cells were cultured for 30min and phosphorylation levels of Akt, mTOR and p70 ribosomal protein S6 kinase (p70S6K) were determined by immunoblotting. Cell proliferation and quantitative polymerase chain reaction assays were conducted using cells cultured for 24h. IGF1 increased phosphorylation of Akt, mTOR and p70S6K and cell proliferation, and these effects were inhibited by inhibitors of IGF1R, Akt and mTOR. Furthermore, IGF1 upregulated the expression of cholesterol biosynthetic genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1) and cytochrome P450, family 5, subfamily A, polypeptide 1 (CYP5A1)), but not sterol regulatory element-binding transcription factor 1 (SREBF1). Increased phosphorylation of p70S6K, a major downstream target of mTOR, and upregulated expression of genes involved in cholesterol biosynthesis are indicative of the key role played by IGF1 in regulating the synthesis of cholesterol, the precursor for steroid hormones.

Testis-Specific Isoform of Na/K-ATPase (ATP1A4) Interactome in Raft and Non-Raft Membrane Fractions from Capacitated Bovine Sperm.

The plasma membrane of sperm contains highly dynamic lipid microdomains (rafts), which house signaling proteins with a role in regulating capacitation. We reported that ATP1A4, the testis-specific isoform of Na/K-ATPase, interacted with caveolin-1, Src, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) in raft and non-raft domains of the plasma membrane of bovine sperm during capacitation. The objective of the present study was to use a proteomic approach to characterize the ATP1A4 interactome in rafts and non-rafts from capacitated bovine sperm. The non-raft interactome included hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein beta1 proteins exclusively, whereas glutathione S-transferase and annexin A2 were unique to raft interactome. However, a disintegrin and metalloprotease 32 (ADAM 32), histone H4, actin, acrosin, serum albumin and plakoglobin were identified in both raft and non-raft fractions of capacitated sperm. Based on gene ontology studies, these differentially interacted proteins were implicated in cell-cell adhesion, signal transduction, fertilization, metabolism, proteolysis and DNA replication, in addition to acting as transport/carrier and cytoskeletal proteins. Overall, we identified proteins not previously reported to interact with ATP1A4; furthermore, we inferred that ATP1A4 may have a role in sperm capacitation.

Testis-specific isoform of angiotensin-converting enzyme (tACE) as a candidate marker for bull fertility.

Although a traditional bull breeding soundness evaluation is designed to identify bulls that are grossly abnormal, bulls classified as satisfactory potential breeders still vary in fertility, implying submicroscopic differences in sperm characteristics. Testis-specific isozyme of angiotensin-converting enzyme (tACE) is involved in the regulation of sperm function. Therefore, the aim of the present study was to determine tACE content, activity and localisation in bull spermatozoa and their associations with fertility. Semen from low-fertility (LF) and high-fertility (HF) Holstein bulls (n=20) with known FERTSOL rates, which represents the 56-day non-return rate, were used. There was greater tACE content (P<0.05) and tACE activity (P<0.01) in HF versus LF spermatozoa. Based on immunolocalisation, tACE was either in the acrosomal or postacrosomal region of the sperm head, with HF bulls having a higher proportion of spermatozoa with tACE in the acrosomal region than LF bulls (P<0.05). tACE content, activity, localisation to the acrosomal region and progressive motility were significantly correlated with fertility and, based on regression analysis, tACE content was predictive of fertility. tACE content and activity in semen were similar between yearling (10-13 months old) and mature (3-4 years old) bulls. Therefore, tACE has potential as a marker of field fertility in bulls at their earliest possible age.

Content and activity of the testis-specific isoform of angiotensin-converting enzyme are reduced in frozen-thawed bull spermatozoa.

Sperm cryopreservation and thawing reduces fertility and alters the content and function of various sperm proteins. Previously, we reported that a testes-specific isoform of angiotensin-converting enzyme (tACE) was required for capacitation of bovine spermatozoa. The aim of the present study was to determine effects of sperm cryopreservation and thawing on the content, activity and localisation of tACE in bovine spermatozoa. Relative median fluorescence intensity (flow cytometry) was greater (P<0.01), tACE content (110 kDa protein) in sperm proteins was higher (P<0.01) and there was greater tACE enzyme activity (mean (±s.e.m.) 0.16±0.01 vs 0.06±0.02UmL-1; P<0.01) in fresh versus frozen-thawed spermatozoa (n=6 bulls). In fresh spermatozoa, tACE was immunolocalised in the acrosomal and principal piece regions of the sperm head and tail respectively. However, in frozen-thawed spermatozoa, there were four patterns of localisation: most frozen-thawed spermatozoa (64%) had fluorescence in the acrosomal ridge, whereas in 17% and 9% of spermatozoa the signal was limited to the post-acrosomal region and the equatorial segment respectively; in the remainder (10%), there was no signal. We conclude that cryopreservation and thawing decrease the content and activity of tACE and cause it to be translocated to other parts of the sperm head.

The ubiquitous isoform of Na/K-ATPase (ATP1A1) regulates junctional proteins, connexin 43 and claudin 11 via Src-EGFR-ERK1/2-CREB pathway in rat Sertoli cells.

Interaction of Na/K-ATPase with its ligand ouabain has been implicated in the regulation of various biological processes. The objective was to investigate roles of Na/K-ATPase isoforms in formation and function of junctional complexes in Sertoli cells. Primary cultures of Sertoli cells were obtained by enzymatic digestion of 20-day-old rat testes and grown on Matrigel-coated dishes for 7 days. Sertoli cells predominantly expressed the ubiquitous isoform of Na/K-ATPase (ATP1A1), confirmed by immunoblotting, PCR, immunofluorescence, and mass spectrometry. Treatment of Sertoli cells with 50 nM ouabain increased transepithelial electrical resistance (TER) and expression of claudin 11 (tight junctions) and connexin 43 (gap junctions), whereas 1 mM ouabain had opposite effects. Involvement of Src-EGFR-ERK1/2-CREB pathway in ouabain-mediated expression of claudin 11 and connexin 43 was evaluated. Incubation of Sertoli cells with 50 nM ouabain increased content of p-Src, p-EGFR, p-ERK1/2, and p-CREB; in contrast, 1 mM ouabain decreased phosphorylation of these signaling molecules. Preincubation of Sertoli cells with inhibitors of Src and MAPK pathways inhibited ouabain-induced effects on these signaling molecules, TER, and expression of claudin 11 and connexin 43. In conclusion, we inferred that ATP1A1 regulated Sertoli cell tight junctions and gap junctions through the Src-EGFR-ERK1/2-CREB pathway. Ouabain is an endogenous steroid; therefore, its interaction with ATP1A1 may be a critical signaling mechanism for the regulation of Sertoli cell function and male fertility.

Content of testis-specific isoform of Na/K-ATPase (ATP1A4) is increased during bovine sperm capacitation through translation in mitochondrial ribosomes.

Capacitation comprises a series of structural and functional modifications of sperm that confer fertilizing ability. We previously reported that the testis-specific isoform of Na/K-ATPase (ATP1A4) regulated bovine sperm capacitation through signaling mechanisms involving kinases. During subsequent investigations to elucidate mechanisms by which ATP1A4 regulates sperm capacitation, we observed that ATP1A4 was localised in both raft and non-raft fractions of the sperm plasma membrane and that its total content was increased in both membrane fractions during capacitation. The objective of the present study was to investigate mechanism(s) of capacitation-associated increase in the content of ATP1A4. Despite the widely accepted dogma of transcriptional/translational quiescence, incubation of sperm with either ouabain (specific ligand for ATP1A4) or heparin increased ATP1A4 content in raft and non-raft sperm membrane fractions, total sperm protein extracts (immunoblotting) and fixed sperm (flow cytometry), with a concurrent increase in Na/K-ATPase enzyme activity. This capacitation-associated increase in ATP1A4 content was partially decreased by chloramphenicol (mitochondrial translation inhibitor) but not affected by actinomycin D (transcription inhibitor). To demonstrate de novo ATP1A4 synthesis, we evaluated incorporation of bodipy conjugated lysine in this protein during capacitation. A partial decrease in bodipy-lysine incorporation occurred in ATP1A4 from sperm capacitated in the presence of chloramphenicol. Therefore, increased ATP1A4 content during capacitation was attributed to mitochondrial translation of ATP1A4 mRNA present in ejaculated sperm, rather than gene transcription. To our knowledge, this is the first report demonstrating ATP1A4 synthesis during bovine sperm capacitation.

Testis-specific isoform of angiotensin-converting enzyme (tACE) is involved in the regulation of bovine sperm capacitation.

We hypothesized that the testis-specific isoform of angiotensin-converting enzyme (tACE) is released during bovine sperm capacitation, and its peptidase activity is required for capacitation. Specific objectives of this study were to (i) develop an anti-tACE antibody; (ii) characterize expression of tACE in bovine testes and sperm; and (iii) determine the role of tACE in capacitation. A 110-kDa protein, consistent with the mass of tACE, was detected in sperm extract by our anti-tACE immunoserum. This immunotarget localized at the acrosomal region and principal piece, but was only expressed in testis of mature bulls. When bull sperm were incubated in Sp-TALP (0 and 4 hr) plus 10 µg/ml heparin (capacitation group) or 10 µg/ml heparin + 10 µM captopril (an ACE inhibitor) for 4 hr, the number of acrosome-reacted (40.1 vs. 24.0%, respectively) and hyperactivated (15.0 vs. 9.7%) sperm increased, and tyrosine phosphoprotein content were higher (p < 0.05) for sperm in heparin alone. tACE activity was also higher (0.04 U/ml; p < 0.01) in incubation medium of sperm exposed to heparin compared to 0- and 4-hr incubation controls or heparin + captopril conditions (0, 0.005, and 0.009 U/ml, respectively). Furthermore, capacitation-associated shedding of a portion of tACE into the medium decreased sperm content of the 110-kDa tACE, but concurrently increased the abundance of a 60-kDa tACE variant. Thus, a portion of the extracellular region of tACE (containing its catalytic site) is released from bovine sperm during capacitation, and tACE activity may be required for sperm capacitation.

Na/K-ATPase regulates bovine sperm capacitation through raft- and non-raft-mediated signaling mechanisms.

Highly dynamic lipid microdomains (rafts) in the sperm plasma membrane contain several signaling proteins that regulate sperm capacitation. Na/K-ATPase isoforms (testis-specific isoform ATP1A4 and ubiquitous isoform ATP1A1) are abundant in bovine sperm plasma membrane. We previously reported that incubation of bovine sperm with ouabain, a specific Na/K-ATPase ligand, induced tyrosine phosphorylation of several sperm proteins during capacitation. The objective of this study was to investigate the roles of lipid rafts and non-rafts in Na/K-ATPase enzyme activity and signaling during bovine sperm capacitation. Content of ATP1A4 and, to a lesser extent, ATP1A1 was increased in raft and non-raft fractions of capacitated sperm, although non-raft enzyme activities of both isoforms were higher than the corresponding activities in rafts from capacitated sperm. Yet, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in both rafts and non-rafts. A comparative increase in phosphorylation of signaling molecules was observed in both raft (CAV1) and non-raft (EGFR and ERK1/2) membrane fractions during capacitation. Although SRC was phosphorylated in both membrane fractions, the non-raft fraction possessed more of this activated form. We also inferred, by immunoprecipitation, that ATP1A4 interacted with CAV1 and EGFR in the raft fraction, whereas interactions of ATP1A4 with SRC, EGFR, and ERK1/2 occurred in the non-raft fraction of ouabain-capacitated sperm; conversely, ATP1A1 interacted only with CAV1 in both fractions of uncapacitated and capacitated sperm. In conclusion, both raft and non-raft cohorts of Na/K-ATPase isoforms contributed to phosphorylation of signaling molecules during bovine sperm capacitation.

A PRELIMINARY ASSESSMENT OF THE EFFECTS OF REPRODUCTIVE SEASONALITY ON SEMINAL PLASMA CHARACTERISTICS IN NORTH AMERICAN BISON (BISON BISON).

The two subspecies of the North American bison, plains ( Bison bison bison) and wood ( Bison bison athabascae) bison, are seasonal breeders. The objective of this study was to conduct a preliminary investigation into the effects of season on semen. To test the hypothesis that there are seasonal effects on seminal plasma, protein profiles of seminal plasma from plains and wood bison (n = 2 of each subspecies) were compared between breeding and nonbreeding seasons. Using two-dimensional gel electrophoresis of seminal plasma proteins, 54 of 170 spots (plains bison) and 19 of 153 spots (wood bison) had differential expression (≥2-fold; P < 0.01) between seasons. Based on immunoblotting, BSP5 and TIMP-2 (two fertility-associated proteins in cattle) were higher during the breeding vs. nonbreeding season. Furthermore, epididymal sperm incubated with seminal plasma from the nonbreeding season had lower postthaw progressive motility (17.33 ± 7.47 vs. 22.09 ± 6.67%; mean ± SD) and an increased ability to undergo a lysophosphatidylcholine-induced acrosome reaction (77.83 ± 8.47 vs. 52.67 ± 7.76%; mean ± SD) as compared to epididymal sperm incubated with seminal plasma from the breeding season. In a heterologous in vitro fertilization system (using bovine oocytes), cleavage rate was higher for sperm exposed to seminal plasma from the breeding vs. the nonbreeding season (75.35 ± 16.55 vs. 33.63 ± 12.44%; mean ± SD). This study suggested that differential expression of seminal plasma characteristics modulating sperm function is one of the mechanisms by which reproductive seasonality affects sperm function in the North American bison.

Enhanced early-life nutrition promotes hormone production and reproductive development in Holstein bulls.

Holstein bull calves often reach artificial insemination centers in suboptimal body condition. Early-life nutrition is reported to increase reproductive performance in beef bulls. The objective was to determine whether early-life nutrition in Holstein bulls had effects similar to those reported in beef bulls. Twenty-six Holstein bull calves were randomly allocated into 3 groups at approximately 1 wk of age to receive a low-, medium-, or high-nutrition diet, based on levels of energy and protein, from 2 to 31 wk of age. Calves were on their respective diets until 31 wk of age, after which they were all fed a medium-nutrition diet. To evaluate secretion profiles and concentrations of blood hormones, a subset of bulls was subjected to intensive blood sampling every 4 wk from 11 to 31 wk of age. Testes of all bulls were measured once a month; once scrotal circumference reached 26cm, semen collection was attempted (by electroejaculation) every 2 wk to confirm puberty. Bulls were maintained until approximately 72 wk of age and then slaughtered at a local abattoir. Testes were recovered and weighed. Bulls fed the high-nutrition diet were younger at puberty (high=324.3 d, low=369.3 d) and had larger testes for the entire experimental period than bulls fed the low-nutrition diet. Bulls fed the high-nutrition diet also had an earlier and more substantial early rise in LH than those fed the low-nutrition diet and had increased concentrations of insulin-like growth factor-I (IGF-I) earlier than the bulls fed the low-nutrition diet. Furthermore, we detected a temporal association between increased IGF-I concentrations and an early LH rise in bulls fed the high-nutrition diet. Therefore, we inferred that IGF-I had a role in regulating the early gonadotropin rise (in particular, LH) and thus reproductive development of Holstein bulls. Overall, these results support our hypothesis that Holstein bull calves fed a high-nutrition diet reach puberty earlier and have larger testes than those fed a low-nutrition diet, and they provide clear evidence that nutritional modulation of Holstein bull calves during early life has profound effects on reproductive development.

KLC3 is involved in sperm tail midpiece formation and sperm function.

Kinesin light chain 3 (KLC3) is the only known kinesin light chain expressed in post-meiotic male germ cells. We have reported that in rat spermatids KLC3 associates with outer dense fibers and mitochondrial sheath. KLC3 is able to bind to mitochondria in vitro and in vivo employing the conserved tetratrico-peptide repeat kinesin light chain motif. The temporal expression and association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the spermatid cell periphery to the developing midpiece suggesting a role in midpiece formation. In fibroblasts, expression of KLC3 results in formation of large KLC3 aggregates close to the nucleus that contain mitochondria. However, the molecular basis of the aggregation of mitochondria by KLC3 and its role in sperm tail midpiece formation are not clear. Here we show that KLC3 expression from an inducible system causes mitochondrial aggregation within 6h in a microtubule dependent manner. We identified the mitochondrial outer membrane porin protein VDAC2 as a KLC3 binding partner. To analyze a role for KLC3 in spermatids we developed a transgenic mouse model in which a KLC3ΔHR mutant protein is specifically expressed in spermatids: this KLC3 mutant protein binds mitochondria and causes aggregate formation, but cannot bind outer dense fibers. Male transgenic mice display significantly reduced reproductive efficiency siring small sized litters. We observed defects in the mitochondrial sheath structure in a number of transgenic spermatids. Transgenic males have a significantly reduced sperm count and produce spermatozoa that exhibit abnormal motility parameters. Our results indicate that KLC3 plays a role during spermiogenesis in the development of the midpiece and in the normal function of spermatozoa.

Angus bulls voluntarily access shade during hot weather, reducing scrotal subcutaneous temperatures and improving sperm quality.

The objective of this study was to determine the relationship of bull location (shade versus no shade), scrotal subcutaneous and ambient temperatures, and sperm quality. Six Angus bulls (4 to 5 y) were randomly allocated into 2 groups of 3 bulls each, housed in 2 outdoor pens, with 1 containing a shed (~3.5 × 6 m and 2.5 m high, 1 open side) to provide shade. Semen was collected by electroejaculation once weekly for 9 wk. The percentage of time a bull voluntarily accessed shade for ≥ 15 min (observed with a game camera) increased with the ambient temperature and ranged from 7.6% to 86.7% for ambient temperatures of < 25°C and > 33°C, respectively. During the 10 hottest days, scrotal subcutaneous temperature (measured hourly with an implanted data logger) in the bulls without access to shade (control group) was directly associated with ambient temperature. Conversely, bulls with access to shade had lower (P = 0.001) scrotal subcutaneous temperatures during high ambient temperatures, particularly when they accessed shade. During the 4 hottest days, these bulls voluntarily accessed shade most of the time from 12:00 noon to 5:00 p.m. (peak ambient temperatures). For total sperm morphological abnormalities and acrosome integrity, there were group effects (P = 0.001 for each), plus a time effect for acrosome integrity (P = 0.009). For total and progressive forward sperm motility, there were group effects (P = 0.001 and 0.023, respectively). For sperm motility kinetics, which were measured with computer-assisted sperm analysis (CASA), [average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), straightness of track (STR), and linearity of track (LIN)], there were also group effects (P = 0.005, 0.011, 0.010, 0.020, and 0.046, respectively). In summary, during hot weather, bulls voluntarily accessed shade, which significantly lowered scrotal subcutaneous temperatures and improved sperm quality.

L'objectif de cette étude était de déterminer la relation entre l'emplacement du taureau (à l'ombre ou sans ombre), les températures sous-cutanées et ambiantes du scrotum et la qualité du sperme. Six taureaux Angus (4 à 5 ans) ont été répartis au hasard en deux groupes de trois taureaux chacun, logés dans deux enclos extérieurs, dont un ayant un abri (~3,5 × 6 m et 2,5 m de haut, un côté ouvert) pour fournir de l'ombre. Le sperme a été recueilli par électroéjaculation une fois par semaine pendant 9 semaines. Le pourcentage de temps pendant lequel un taureau a accédé volontairement à l'ombre pendant ≥ 15 min (observé avec une caméra de chasse) augmentait avec la température ambiante et variait de 7,6 % à 86,7 % pour des températures ambiantes de < 25 °C et > 33 °C, respectivement. Pendant les 10 jours les plus chauds, la température sous-cutanée scrotale (mesurée toutes les heures avec un enregistreur de données implanté) chez les taureaux sans accès à l'ombre (groupe témoin) était directement associée à la température ambiante. À l'inverse, les taureaux ayant accès à l'ombre avaient des températures sous-cutanées scrotales plus basses (P = 0,001) lors de températures ambiantes élevées, en particulier lorsqu'ils accédaient à l'ombre. Durant les quatre jours les plus chauds, ces taureaux ont accédé volontairement à l'ombre la plupart du temps de 12h00 à 17h00. (températures ambiantes maximales). Pour les anomalies morphologiques totales des spermatozoïdes et l'intégrité de l'acrosome, il y avait des effets de groupe (P = 0,001 pour chacun), plus un effet de temps pour l'intégrité de l'acrosome (P = 0,009). Pour la motilité totale et progressive des spermatozoïdes, il y avait des effets de groupe (P = 0,001 et 0,023, respectivement). Pour la cinétique de la motilité des spermatozoïdes, qui ont été mesurées avec une analyse de sperme assistée par ordinateur (CASA), [la vitesse moyenne du trajet (VAP), la vitesse curviligne (VCL), la vitesse en ligne droite (VSL), la rectitude de la voie (STR) et la linéarité de la voie (LIN)], il y avait aussi des effets de groupe (P = 0,005, 0,011, 0,010, 0,020 et 0,046, respectivement). En résumé, par temps chaud, les taureaux ont volontairement accédé à l'ombre, ce qui a considérablement abaissé les températures sous-cutanées du scrotum et amélioré la qualité du sperme.(Traduit par Docteur Serge Messier).